This is a revised application from a highly productive, multidisciplinary, multicenter project evaluating the diagnosis of women at risk for ectopic pregnancy (EP) and early pregnancy failure. Early pregnancy failure is the most common complication in pregnancy with an estimated 25% of clinically recognized pregnancies ending in miscarriage, and 1-2% are diagnosed as ectopic pregnancy (EP). EP is the leading pregnancy-related cause of death and a major contributor to maternal morbidity. When the gestation is not visible with ultrasound, distinguishing a healthy ongoing intrauterine gestation (IUP) from a miscarriage or an EP poses a critical clinical challenge as there is no other definitive, noninvasive diagnostic test. Current diagnostic protocols can result in the interruption of a desired IUP and/or morbidity associated with rupture of an EP. We have demonstrated that a number of putative biomarkers of EP can distinguish IUP from EP when used in combination. Using an unbiased proteomic analysis approach we have also established new biomarkers, one which we have already validated: ADAM-12. The goal of this project is to expand the use of biomarkers to develop a biosignature distinguishing EP, ongoing IUP and miscarriage. Specific Aims and Methods: We plan to develop a multiple marker bio-signature profile to aid in the diagnosis of EP. We hypothesize that a small number of markers, used in combination, will be able to distinguish an EP from IUP (combination of ongoing IUP and miscarriage) and/or can be used to distinguish a nonviable gestation (combination of EP and miscarriage) from an ongoing IUP. In Specific aim 1 we will develop, refine and validate a serum (multiple marker) test to identify EP based on putative markers of early implantation and viability. In Specific Aim 2 we will validate the potential of newly discovered biomarkers of EP to add to (or replace) those in our current multiple marker panel. In Specific aim 3 we will conduct an unbiased three-way proteomic discovery study that will identify new, lower abundance biomarker candidates that distinguish between EP, IUP and miscarriage. Summary: This proposal represents a unique opportunity to combine epidemiologic and basic science methodologies to understand and improve upon important limitations in our ability to diagnose and treat a reproductive disorder with important public health consequences. Utilizing access to a large number of geographically, racially and ethnically diverse women at risk for EP, we plan to conduct a series of multicenter case control studies using well phenotyped bio-specimens, and ultimately conduct a prospective cohort study to assess actual use in the intended patient population. The development of non invasive methods of diagnosis, such as a serum test to diagnose an EP and/or miscarriage with high accuracy, would have tremendous clinical impact.